1. Field of Invention
This invention relates to electrophoresis equipment and method, and specifically to improved vertical slab gel electrophoresis equipment and related methods.
2. Prior Art and Comment
Ferdinand Ruess, a Russian physicist, watched the migration of clay colloidal particles between two electrodes in 1897. About 50 years later, Arne Tiselius, a Swedish chemist, studied the migration of protein molecules in electric field, demonstrated the complex nature of serum proteins by using a prototype that he termed as a free electrophoresis apparatus. Whereby he won the Nobel Prize for chemistry of 1948. Electrophoresis has now become a versatile and powerful technique in biomedical and related research areas. It can be employed to fractionalize almost any charged particle, from ionizable small molecules to whole cells. When an electrophoresis carries out in a gel matrix, it terms as gel electrophoresis. Gel electrophoresis has very high resolution, is mainly employed to fractionate biomacromolecular species, such as DNA, RNA or proteins. That is because the network of the gel matrix acts as a molecular sieve to retard the migration of the macromolecular species according to their size and shape. Besides, the gel matrix can also stabilize the boundaries of the separated species both during and after the electrophoresis, so as to facilitate the subsequent analyses. The widely employed gel matrixes are agarose gel and polyacrylamide gel. The latter can be cast into a vertical cavity due to it can adhere better to the cavity walls. By successfully exploited the high fractionation ability of vertical slab polyacrylamide gel electrophoresis for determination the base sequences of DNA, Frederick Sanger, a British biochemist, and his American colleague Walter Gilbert were awarded the Nobel Prize for chemistry of 1980.
In vertical slab gel electrophoresis (abbreviated as VSGE hereafter), the electrophoretic vector travels vertically within a slab shaped gel matrix uprightly arranged between an upper and a lower pH buffer solution chambers charged with opposite electrodes. Usually the gel slab is only 0.2 to 2.0 mm in thickness. None such a gel slab can stand uprightly by itself unless it is held in a cassette; none such a gel slab can be properly held in a cassette unless it is directly cast and formed in it. Therefore, and obviously, there are three basic problems regarding the design of any VSGE cell: (i) how to construct a vertical slab gel casting cassette (abbreviated as VSGC cassette, gel casting cassette, or cassette hereafter) having sealed left and right edges but opened up and low ends; (ii) how to insure a gel matrix to be cast into the cassette without leakage; and (iii) how to urge the cassette to water-tightly join the upper buffer chamber (abbreviated as UBC hereafter) with the upper opening of the cassette exposes into the formed cassette/UBC complex. As long as the cassette/UBC complex forms, just sets it into a lower buffer chamber (abbreviated as LBC thereafter), thereupon a VSGE cell is accomplished. As for how to arrange the electrodes in the UBC and LBC, and how to make a lid for the VSGE cell, those are foolproof.
About the size: Typically there were three different sized VSGE cells. The 36×30 cm slab gels were usually used for DNA sequencing electrophoresis. But it has been replaced by some automatic capillary gel electrophoresis instruments in the developed countries, referring to U.S. Pat. No. 5,374,527 (1994), etc. The 18×16 cm slab gels were widely used for protein fractionation in the early years. However nowadays, more than 95% chance is to run the mini gel, which casts in 8×10, or 10×10 cm cassettes, due to its shorter running time and easer to manipulate.
About the structure style: There were several different styled VSGE cells, referring to U.S. Pat. No. 3,719,580 (1973), U.S. Pat. No. 4,224,134 (1980) and U.S. Pat. No. 4,574,040 (1986), etc. However, the most popular styled VSGE cell is a kind of dual gel cell, which was initiated by Madjar et al (1) in 1977. Thereafter various VSGE cells were patented, but most of them wore still belong to the most popular styled dual gel cell as mentioned above, referring to U.S. Pat. No. 4,574,040 (1986), U.S. Pat. No. 5,632,877 (1997), U.S. Pat. Nos. 5,888,369 and 6,001,233 (1999), etc. Their common structure style is that; on one hand, making the UBC to have two opposite U-shaped side openings; on the other hand, making each cassette to have a U-notched upper opening; and then using an urging mechanism to force those two cassettes to sandwich that UBC between them. Certainly, U-shaped rubber sealing-gaskets are always employed for sealing up the interfaces between the cassettes and UBC. As a result, a water-tightly joined cassettes/UBC complex is formed with the two U-notched upper openings naturally expose into the formed complex. However, their common weakness is that when only one gel runs in such a dual gel cell, the other side opening of the UBC has to be blocked up. It is inconvenient, because more than 50% chance is to run one gel a time. In CN Pat. 88106198.0 (1982), the inventor developed a modular VSGE cell, which allows numerous slab gels to run in it parallel.
About the cassette: compare with any kind plastic, glass plate is a better material to wall VSGC cassettes, due to it has much higher rigidity, much higher chemical inertness, much higher thermal conductivity; and especially due to the gel matrix can adhere it better. Ceramic, such as aluminum oxide plate, is even better than glass, but it is not transparent and cannot be as cheap as glass plate. Unfavorably, both of them are typical bad machining-able materials, so that to make glass and/or ceramic walled cassettes can never be as easy as to make them by plastics. Nevertheless, any reusable cassette is had better to be glass and/or ceramic walled by the reason as mentioned above. The simplest glass walled cassettes was formed by two identical rectangular glass plates and a pair of flat plastic spacer strip, as reported by Herbert Tichy (2) in 1966. However, this kind cassette is not easy to join the UBC, and its sample loading area is not easy to access, referring to U.S. Pat. No. 4,224,134 (1980). Afterward, one of the two rectangular glass plates was replaced by a U-notched glass plate, so as to make the cassette having a U-notched upper opening, referring to the report of F. W. Studier (3) in 1973. However, the U-notched glass or ceramic plates are much more costly and much more fragile than rectangular plates. Subsequently, the U-notched glass wall was replaced back by a shorter rectangular glass wall, as in U.S. Pat. No. 4,574,040. Although the shorter glass wall along with two flat plastic spacer strips also can form a U-notched upper opening for the cassette, but this way formed U-notched upper opening does not have an even rim. As a result, the leakage of the upper pH buffer solution becomes the major problem if this kind cassette is employed in any VSGE cell, referring to the Tech Note (4). Cross section T-shaped spacer strips were used to form a cassette in U.S. Pat. No. 4,560,459 (1985), but that cassette still had to use a U-notched sidewall. A three-element cassette was disclosed in U.S. Pat. No. 4,954,236 (1990), but its abutting face has no even margin to insure a leak-free abutting.
Besides, a fact was examined in the prior art. That is under appropriate pressure, the left and right margins of those glass walled VSGE cassettes could achieve leak-free, provided the employed two plastic spacer strips are wide and smooth enough, therefore makes no need using grease or glue to seal up the left and right margins.
About the urging mechanism: An urging mechanism is always required for forcing the cassette and the UBC to rest on each other tightly. Most employed urging mechanisms were too complex and lax, so that large LBC were always required, refereeing to U.S. Pat. No. 4,574,040 (1986), U.S. Pat. No. 5,632,877 (1997) and U.S. Pat. Nos. 5,888,369, 6,001,233 (1999), etc. A compact clamp urging mechanism was disclosed recently in U.S. Pat. No. 6,436,262 (2002), but it looks short of compatibility.
About the gel casting: Essentially the process of gel casting is same to the process of the Plexiglas plate manufacturing, as disclosed in U.S. Pat. No. 2,154,639 (1939) of Rohm et al. However, herein the formed polymer is a hydrophilic gel matrix, is not supposed to be moved out off the mold for any other use, but is for stay in situ as a matrix for an electrophoresis to take place therein. Most ordinary VSGC cassettes can be arranged in face to face, put into a gel-casting box to perform gel casting. The first gel casting box, and the method of gradient slab gel casting was reported by Margolis et al (5) in 1968. Different size, different improved gel casting boxes are commercially available nowadays. However, their common defect is lacking of flexibility. G. P. Magnant patented a casting method for forming a gel matrix in U.S. Pat. No. 5,188,790 (1993). However since several thousand years ago, our human being already knew how to form objects by casting. The chemical mechanism of the polyacrylamide gel formation for electrophoresis was published by Leonard Ornstein (6) in 1964. Of course, any kind of casting needs a mold. If Magnant patented apparatuses is a three-element assembled mold, it had been disclosed by Rohm et al 54 years ago before him. Even though, the problem is how to carry out an electrophoresis in such a three-element mold, which has no lower opening. Overlooking of all other problems, and if there is no misunderstood to us, then the patented method of Magnate seems nothing more than putting a ordinary cassette into a ordinary loose plastic membrane bag, and then using four objects from four sides to push the loose bag towards the cassette for gel casting. If so, Magnant patented method looks neither convenient nor flexible than using those gel casting boxes. Several different methods were designed to seal up the lower openings of VSGC cassettes for gel casting, referring to U.S. Pat. No. 4,224,134 (1980), U.S. Pat. No. 5,192,408 (1993), U.S. Pat. No. 5,520,790 (1996), U.S. Pat. Nos. 6,110,340 and 6,162,342 (2000), etc. But most of them have no compatibility, some are not dependable, some are inconvenient.
About the heat absorbing device: Joule-heating generates in the gel matrix during electrophoresis. A heat-absorbing device is required in a VSGE cell when the samples need to run in native state. However in most cases, such as in DNA sequencing gel electrophoresis or SDS protein gel electrophoresis, the samples need to run in denatured state, therefore making no heat-absorbing device is required. All heat-absorbing devices in the prior art need to use exogenous coolant, referring to U.S. Pat. No. 4,224,134 (1980) and U.S. Pat. No. 4,574,040 (1986), etc.